Chemical pesticides have been used intensively to control pests for many years. An awareness of recent problems in the use of pesticides and concern about their adverse effects on man and his environment have resulted in more commercial attention being given to biological control alternatives. Certain entomogenic fungi have been recognized by researchers to be pathogenic to different pests; particularly the use of entomogenic fungi has been widely studied as biological control agents in the USSR and in Europe. Update reviews on the different pathogenic fungi and their use and status can be found in: Carlo M. Ignoffo and B. Handava "Handbook of Natural Pesticides", vol. V, part A., C. W. McCoy et al. "Microbial Insecticide", CRC Press, 1988, and M. N. Burge "Fungi in Biological Control Systems", Manchester University Press, 1988. Unlike insect pathogenic bacteria or other microorganisms (e.g., viruses or protozoa) which must be ingested by the insect to initiate diseases, entomogenic fungi normally invade through the host's cuticle.
Entomogenic fungi infect pests, usually insects, by a parasitism disease mechanism. The infection process development is believed to consist of the following steps:
1. Attachment--The conidium of the entomogenic fungi spore is attached to the insect cuticle. PA0 2. Germination--The conidia spore is germinated on the insect cuticle to form a germ tube. PA0 3. Penetration--The germ tube penetrates directly into the cuticle. It is believed that the cuticular invasion involves both enzymatic and physical activities. PA0 4. Growth--The fungus grows in the hemocoel as mycelium or blastospore. The fungi overcome the host by invasion of organs. PA0 5. Saprophytic Growth--The fungi grows on the outside of the insect and produces aerial conidia spores. PA0 Stage I: Production of fungal biomass in very high yield in fermenter in the form of filamentous mycelium. PA0 Stage II: Harvesting the filamentous mycelium, formulating the wet mycelium, and encapsulating the formulated wet mycelium in calcium alginate prills. PA0 Stage III: Drying the formulated prills and packaging the product for use as a biopesticidal agent. PA0 Stage IV: Activating and cultivating the prill to produce pathogenic conidia spores. PA0 Stage V: Drying the reactivated prill and packaging the product for use as a biopesticidal agent. PA0 Stage VI: Harvesting the pathogenic conidia spores from the reactivated prill and using the conidia spores as a biopesticidal agent. PA0 Stage VII: Pregermination of the pathogenic conidia spores harvested from the reactivated prill and using the pregerminated conidia spores as a biopesticidal agent.
Some entomogenic fungi overcome their host before extensive invasion of organs takes place, presumably by production of toxins. Although toxic compounds have been reported from culture filtrate of mycelium of several entomogenic fungi (e.g., Paecilomyces fumosoroseus was shown to produce the toxin Beauvericin (Ferron, Annual Review of Entomology 23:409-442 (1978), and a peptide toxin known as destruxin A has been isolated from culture broth of Metarrhizium anisoplia (Y. Kodaira, Agr. Biol. Chem. Chem. 26-36 (1962)), only in few cases it was reported that toxins have been detected in insects infected with the entomogenic fungi (Suzuki et al., Agric. Biol. Chem. 35:1641-1643 (1971)).
The use of the entomogenic fungi for control of different pests is not itself a new idea. Entomogenic fungi such as Metarrhizium, Beauveria, Hirsutella, Verticillium or Paecilomyces have been studied for development as pest control agents. Solid state fermentation has been widely examined because this method allows the production of the infectious bodies of the entomogenic fungi, e.g., conidia spores. However, it has been found that conidia spores of the various entomogenic fungi are very sensitive to drying processes and the conidia spores lose their viability very quickly.
Submerged fermentation have significant problems to overcome. Most of the entomogenic fungi heretofore grown in submerged culture produce mostly blastospores with some mycelium. Blastospores are not stable during storage or drying processes. Attempts to formulate blastospores resulted in reduced efficacy and stability.
In order to overcome the above stability problems associated with conidia spores and blastospore preparations, several processes have been disclosed in the literature for the production of biocontrol agents based on biomass obtained from submerged culture fermentation. These techniques generally involve growing fungi in liquid media followed by inoculation of a solid media or an inert carrier, such as vermiculite, on which the conidia spores are produced. For example, Kybal (1976) discloses a process for incubating biomass containing mycelium, blastospores and other fungal stages in shallow, aerated vessels to produce conidia spores directly on the surface of the coated vessel surface. However, this process is very labor intensive and the conidia spores obtained is only on the vessel surface. The surface area per apparent volume tends to be small and the efficiency is low.
In another process McCabe et al., (U.S. Pat. No. 4,530,834), discloses the use of entomophthoralean mycelium produced in submerged culture for the production of resting conidia spores. This process is a diphasic system whereby production of spores or blastospores is bypassed. First, the biomass produced by liquid fermentation is dried with protective agents, and the dry biomass mat obtained is milled to a dry powder form. Next, the dry powder preparation is rewetted and applied to the target pest. However, this method is plagued with stability problems. The powder product needs to be stored below 4.degree. C. to maintain its viability. Even under such storage conditions the product is only stable for 64 days. Further, conidia spores produced from reconstituted powder are short lived and fragile.
Dried mycelium particles obtained from liquid fermentation of different entomogenic fungi have been produced in similar ways by different investigators. For example, McDonald et al. (Vth International Colloquium on Invertebrate Pathology and Microbial Control, Adelaide, Aus, p. 147) disclose a similar basic process for the entomogenic fungus Culicinomyces clavisporus for mosquito control. This process involves growing the fungus in liquid culture for 6 days, harvesting the suspension by filtration and adding sucrose followed by air drying. The dried mycelial mat was then ground in a hammer mill and sieved through a 3.5 .mu.m sieve. Upon addition of these mycelium particles to water approximately 5.times.10.sup.6 conidia spore/mg of dried particle were produced. Dried mycelium particles could be stored for 1-5 weeks at 4.degree. C. without losing activity. A similar approach has been used by Roberts et al. (Vth International Colloquium on Invertebrate Pathology and Microbial Control, Adelaide, Aus, p. 336) for the use of dried mycelium particles of Metarrhizium anisopliae isolate ARSEF 2457 to control the Japanese beetle and other pests. Based on the Roberts et al. results it has been found that lyophilized Metarrhizium mycelium has a longer shelf life in soil than conidia spores.
Bayer A. G. (EP application 0268117 A2, 1987) has reported on a similar method which is based on the production of mycelium and blastospores of Metarrhizium anisopliae in a fermenter. During the course of fermentation, the mycelium/blastospore aggregate to form pellets in the size of 0.1 mm up to 1.5 mm in diameter. At the end of the submerged culture fermentation, the mycelium/blastospore aggregates are harvested and dried in a fluidized bed dryer to form a final product which consists of dry mycelium/blastospore granules with 0.5 to 1.5 mm diameters. The granules can be applied to soil where they can form infectious conidia spores. However, this method suffers from several problems. First, the fungal pellets formed in the fermenter result in a very low biomass yield. Therefore, such a fermentation process is not economical. Also, the final granular product obtained must be stored under vacuum and at a low temperature. It seems also that the conidia spores formed after activation in the soil have a short life. This is probably because the granules do not contain any nutrients which can stimulate or promote growth.
Another approach, which has been used widely for the delivery of different fungal pathogens (mainly conidia spores and chlamydospores) against plant diseases, is encapsulating fungal spores in alginate prills (Lewis et al., Proceed. Intern. Symp. Control. Recs. Bioact. Mater, 12:341-3 (1985), Fravel et al., Phytopathology, 27:3341-8 (1982), and J. J. Morois, U.S. Pat. No. 4,724,147). This method has been ignored in general for the delivery of entomogenic fungi for insect and other pest control. Few publications on the use of alginate prill for delivery of entomogenic fungi are known. For example, Enrique A. Cabanillar (Ph.D. thesis) "Factors Influencing the Efficacy of Paecilomyces lilacinus in Biocontrol", 1987, North Carolina State University, described this method to deliver conidia spores of Pacilomyces lilacinus produced on solid media (solid state fermentation) against Koot-knot nematode. Roberts, et al. (Entomopathogenic Fungi: Recent Basic & Applied Research; Matha, V. et al. (ed) Biopesticides Theory & Practice Proc. Conf., Sep. 25-28, 1989, Ceske, Budejovice, Czechoslovachia 11-30 pp) disclose a process for using alginate pellets which incorporate the mycelium biomass of Beaveria bassiana & Metarrhizium anisopliae with pre-gelatinized starch as a basic nitrogen supply.